The present invention relates to methods of isolating chondrocytes, methods of culturing isolated chondrocytes to generate cultured chondrocytes/bone cells, and to uses of such cultured chondrocytes/bone cells. More particularly, the present invention relates to methods of isolating mandibular condyle chondrocytes, methods of culturing same to generate highly differentiated cultured chondrocytes/endochondral bone cells, and to uses of such cultured chondrocytes/endochondral bone cells for treating cartilage/bone diseases.
Cartilage/bone diseases include highly debilitating and/or lethal diseases such as arthritis, articular cartilage injury, meniscal disorders, joint infections, chondrogenesis disorders and cosmetic disorders of cartilaginous structures of the body for which no optimal therapies are currently available. Failure of diarthrodial joints leads to arthritis, the most common form being osteoarthritis. Repair of arthritic joints requires orthopedic surgery to replace the worn-out joints by a prosthesis or by a biological graft. Arthritis alone is an enormous medical and economic problem, with more than thirty million Americans suffering from this highly debilitating disease.
Articular joints, of which various types exist in the body, are a vital component of the musculoskeletal system. Freely moving joints (ankle, elbow, hip, knee, shoulder, and those of the fingers, toes and wrist) are known as diarthrodial or synovial joints and are critical for body movement. The intervertebral joints of the spine, which are not diarthrodial joints as they are fibrous and static, critically provide the flexibility required by the spine. Diarthrodial joints enable local motion and other activities of daily life to take place. They perform their function so well that we are often not even aware of their existence nor the function they provide until injury strikes or arthritis develops. From an engineering point of view, these natural bearings are very uncommon structures. Under healthy and ideal conditions, their function is nearly frictionless and they remain almost entirely wear-resistant throughout life. Diarthrodial joints share common structural features, notably including their being enclosed in a strong fibrous capsule. The inner surfaces of the joint capsule are lined with a metabolically active tissue, the synovium, which secretes the synovial fluid that lubricates the joint and provides the nutrients required by the avascular cartilage. The articulating bone ends in the joint are lined with a thin layer of hydrated soft tissue known as articular cartilage. These linings, the synovium and articular cartilage layers, form the joint cavity which contains the synovial fluid. Thus, in animal joints, the synovial fluid, articular cartilage, and the supporting bone form the bearing system which provides the smooth nearly-frictionless bearing system of the body. While diarthrodial joints are subjected to an enormous range of loading conditions under cyclical conditions, the cartilage surfaces undergo little wear and tear under normal circumstances. Indeed, most human joints must be capable of functioning effectively under very high loads and stresses and at very low operating speeds. These performance characteristics demand efficient lubrication processes to minimize friction and wear of cartilage in the joint. The joint is stabilized by, and in motion is controlled by, ligaments and tendons which may be inside or outside the joint capsule. Breakdown of the joint cartilage as a result of autoimmune, physical, chemical and/or infectious insult leads to arthritis.
Hyaline cartilage, the most abundant form of cartilage, is glass smooth, glistening and bluish white in appearance, although older or diseased tissue tends to lose this appearance. The most common hyaline cartilage, and the most studied, is the articular cartilage. This tissue covers the articulating surfaces of bones within diarthrodial joints. Articular cartilage is characterized by a particular structural organization, consisting of specialized cartilage cells termed “chondrocytes” embedded in an intercellular material, typically referred to as “cartilage matrix”, which is rich in proteoglycans, collagen fibrils, other proteins, and water. While cartilage tissue is neither innervated nor penetrated by the vascular or lymphatic systems, in the mature joint of adults, the underlying subchondral bone tissue—which forms a narrow, continuous plate between the bone tissue and the cartilage—is innervated and vascularized. Beneath this bone plate, the bone tissue forms trabeculae, containing the marrow. In immature joints, articular cartilage is underlined by only primary bone trabeculae. A portion of the meniscal tissue in joints (referred to as the “interarticular” cartilage) also consists of cartilage whose make-up is similar to articular cartilage. It is generally believed that because articular cartilage lacks a vasculature, damaged cartilage tissue does not receive sufficient or proper stimuli to elicit a repair response.
The menisci of the knee, and other similar structures such as the disc of the temporomandibular joint and the labrum of the shoulder, are specialized fibrocartilagenous structures which perform functions which are vital for normal joint function. They are known to function in assisting the articular cartilage in distributing loads across the joint, assisting the ligaments and tendons in providing joint stability, playing a major role in shock absorption, and possibly in assisting lubrication of the joint. The menisci, disc and labrum are hydrated fibrocartilage structures composed primarily of collagen (type I) with smaller amounts of other collagens and proteoglycans (including aggrecan and the smaller, non-aggregating proteoglycans). They contain a sparse population of cells which, like the chondrocytes of cartilage, are responsible for the synthesis and maintenance of this extracellular matrix. Damage to these structures can lead to a reduction in joint function and degeneration of the articular cartilage, and surgical removal of such damaged structures, usually the main treatment, can result in early onset of osteoarthritis.
Skeletal ligaments are specialized connective tissues that connect bones. They serve a passive mechanical function in stabilizing joints and in guiding joint motion. Further, they may have a neurosensory role transporting dynamic information to muscles. Ligaments are composed primarily of type I collagen organized in parallel arrays, with small amounts of other collagens, proteoglycans, elastin and other proteins and glycoproteins. The cells are fibroblastic in the midsubstance, and appear more chondroid at and near the insertion sites. Tendons have a similar structure, with a relatively high concentration of collagen, organized primarily as fibers in parallel. Other components are proteoglycans, elastin and other proteins and glycoproteins. The cells are fibroblastic in nature. The cells of the tendon and ligament are metabolically active and are responsible for the synthesis and maintenance of this extracellular matrix.
Proteoglycans comprise the second largest portion of the organic material in articular cartilage. These macromolecules are composed of a protein core to which are attached a number of covalently bound glucosaminoglycan (GAG) chains, such as chondroitin sulfate and keratan sulfate. There are many different types of proteoglycans present in a wide range of tissues throughout the body; presumably, they also have different functions in the various tissues. However, the most extensively studied proteoglycans have been those from articular cartilage because of their role in regulating skeletal growth, joint function and the development of arthritis.
The major proteoglycans present in articular cartilage are the large aggregating type (50-85%) and the large non-aggregating type (10-40%) with distinct small proteoglycans also present. The molecular weights of these proteoglycan monomers range from 1,000-4,000 kDa, and they contribute significantly to the mechanical and physicochemical properties of cartilage. These molecules are highly ordered structures with length scales ranging from 0.01-1 microns. Proteoglycans comprise an extended protein core with several distinct regions, including an N-terminal region with two globular domains (G1 and G2), a keratan sulfate-rich domain, a longer chondroitin sulfate-rich domain which may also contain some interspersed keratan sulfate and neutral oligosaccharide chains, and a C-terminal globular domain (G3) on the proteoglycan monomer. Aggregates are formed when many proteoglycan monomers bind to a long monofilament chain of hyaluronan via their G1 globular domain. Each proteoglycan-hyaluronan bond is stabilized by a separate 41-48 kDa globular link protein. The structure of proteoglycan in cartilage is not uniform. Differences in chain length and amounts of keratan sulfate and chondroitin sulfate, length of the protein core, and degree of aggregation all contribute to the compositional and structural heterogeneities of proteoglycans within cartilage.
The GAG chains of the proteoglycans afford important physicochemical properties to cartilage. First, chondroitin sulfate which has a molecular weight of about 20 kDa is composed of repeating disaccharide units of glucuronic acid and N-acetylgalactosamine with one sulfate (SO4) group and one carboxyl (COOH) group per disaccharide. Evidence exists indicating that these chondroitin sulfate chains are the main determinants of frictional resistance against interstitial fluid flow. Keratan sulfate consists of repeating disaccharide units of galactose and N-acetylglucosamine, again averaging approximately one sulfate group per disaccharide. The keratan sulfate content of proteoglycans progressively increases with age from fetal to senescent cartilage. Both proteoglycan content and size decrease with increasing severity of osteoarthritis.
In articular cartilage, molecular interactions occur through collagen-collagen covalent cross-link interactions, and proteoglycan-proteoglycan and collagen-proteoglycan non-covalent (electrostatic and mechanical) interactions. The best-known interactions are the collagen-collagen covalent cross-links which are important in providing a strong and stiff collagen network. Thus, in the extracellular matrix these two molecular networks (proteoglycan and collagen) must coexist to form a fiber-reinforced composite solid with the collagen network providing the tensile stiffness and strength, and the proteoglycan network providing the compressive stiffness. The physical interactions between collagen and proteoglycan can arise from two sources: electrostatic and mechanical. First, evidence exists indicating that the negative charge groups on the proteoglycans can interact with the positive charge groups along the collagen fibrils, and hyaluronates of the aggregate do interact with type II, IX and X collagen. Second, evidence of strong frictional interaction between the proteoglycans and the fine collagen network also exists. No covalent bonding exists between collagen fibrils and proteoglycans. In normal cartilaginous tissue, proteoglycans are slowly but continuously turned over, the degraded molecules are released from the cartilage and are replaced by newly synthesized components. It is the coordinate control of synthesis and degradation of the matrix components by the chondrocytes that maintain normal cartilage. In experimental models of joint disease, for example, there is evidence of charges in the rate of biosynthesis and turnover of proteoglycans, which may contribute to cartilage degeneration. This chondrocyte-mediated degeneration leads to the whole cascade of degenerative bone and connective tissue events that results in osteoarthritis, limb immobilization, and other effects requiring surgical intervention. Degenerative loss of articular cartilage, for example, at the acetabular/femoral head articulation, results in heavy loading of the soft tissue, and can require radical surgery.
Chondrogenesis is vital to postnatal skeletal growth which occurs mainly by endochondral bone formation, a highly regulated multistep process. The skeletal cellular population follows a cascade of events that includes proliferation of precursor cells, differentiation into chondroblasts, maturation of chondrocytes, hypertrophy, and apoptosis [Chen, Q. et al., 1995. Dev Biol. (N.Y. 1985) 172:293-309]. These processes are accompanied by the synthesis of specific matrix proteins such as cartilage proteoglycans and type II collagen, which are secreted by mature chondrocytes, and type X collagen, which is secreted by hypertrophic chondrocytes (Beier, F. et al., 1999. J Cell Biochem 72:549-557). The sustenance of the differentiated state of the chondrocytes is dependent on close cell-matrix interactions (Svoboda, K. K., 1998. Microsc Res Tech. 43:111-122), such that releasing the cells from their cartilaginous environment results in a rapid loss of their phenotypic morphology and function (von der Mark, K. et al., 1977. Nature 267:531-532). Normal chondrogenesis is a complex process controlled by a combination of systemic and local factors such as growth hormone, thyroid and parathyroid hormones, and, during various developmental stages, also by insulin and sex hormones, the neonatal period and adolescence, respectively (Amizuka, N. et al., 1994. J. Cell Biol. 126:1611-1623; Greenspan, S. L. and Greenspan, F. S., 1999. Ann Intern Med 130:750-758; Maor, G. et al., 1999. Endocrinology 140:1901-1910; Menon, R. K. and Sperling, M. A., 1996. Endocrinol Metab Clin North Am 25(3):633-647; Spagnoli, A. and Rosenfeld, R. G., 1996. Endocrinol Metab Clin North Am 25:615-631). Insulin-like growth factor-I (IGF-I) is the principal local growth factor of chondrogenesis and skeletal growth and acts in an auto/paracrine fashion (Isgaard, J., 1992. Growth Regul 2:16-22).
As described hereinabove, cartilage/bone diseases are of tremendous medical and economic impact, and hence there is an obvious and urgent need for novel and improved methods of treating such diseases.
For example, over one million surgical procedures in the United States each year involve cartilage replacement. Current therapies include transplantation with allografts (removing healthy cartilage from a donor, and reimplanting it into a joint of the recipient), implantation of artificial polymer or metal prostheses, and surgical removal of old or degenerative cartilage and the surgical treatment of underlying bone to stimulate new cartilage formation. This new cartilage is usually a fibrous cartilage significantly inferior to the hyaline cartilage it is replacing. Other surgical procedures of synovial joints involve the replacement of menisci, ligaments and tendons with biological grafts or artificial tissues. Torn or severed menisci, discs of the temporomandibular joint, labrum of the shoulder, tendons and ligaments often undergo surgical repair.
Surgical procedures account for only a fraction of the treatment of individuals who suffer from disabling diseases resulting from connective tissue damage and degeneration in synovial joint. Alternative treatment includes conservative treatment (e.g., rest and physical therapy), and treatment is largely directed at symptomatic relief through the use of analgesics and nonsteroidal anti-inflammatory drugs.
There are significant limitations with all present approaches. Artificial prostheses have a limited lifetime, and can fail prematurely. Recurrent replacements of prostheses is not an advisable treatment, and, therefore, the relatively young and active patient is often consigned to slow joint degeneration until the use of prosthetic implants becomes a reasonable clinical option. Prostheses rarely replicate the performance of the original tissue. A prosthesis cannot adapt in response to environmental stress as does a biological tissue, nor can it repair itself. Biological allograft material is in limited supply, appropriate size shape and tissue type are difficult to obtain, and has the risk of carrying infectious diseases. Use of autograft material compromises the site used for the source tissue (e.g., using patella tendon to replace anterior cruciate ligament), and can only offer this tissue once.
In light of the above described drawbacks of classical treatment methods, an optimal strategy for treating cartilage/bone diseases, would be to utilize cultured cartilage/bone to repair or replace cartilage/bone lost or damaged as a result of disease or injury. Such an approach would be optimal since cultured cartilage/bone could theoretically be produced with essentially any desired characteristics and in essentially any desired quantity. However, culturing cartilage/bone from primary chondrocytes has been found to be highly problematic due to the fact that primary cartilage-derived cell cultures undergo dedifferentiation, acquire fibroblastic features, and lose most of the characteristics of mature chondrocytes. This phenomenon is due mainly to the loss in culture of the close matrix-cell interrelationship typical of cartilage tissue, which, as described above, is a vital element of cartilage formation and homeostasis. This dedifferentiation phenomenon is furthermore a serious obstacle for ex-vivo studies of the endochondral ossification process and its extra- and intracellular regulation, and for in-vitro studies of various articular pathologies such as rheumatoid arthritis or osteoarthritis.
Several prior art approaches have been employed or suggested in order to optimally generate cultured cartilage/bone.
One approach involves culturing limb mesenchyme in micromass cultures in three-dimensional collagen and agarose gels (Miura, T. and Shiota, K., 2000. Anat Rec. 258:100-107).
Another approach involves culturing isolated cells of mouse limb bud mesenchyme (Shakibaei, M. and De Souza, P., 1997. Cell Biol Int 21:75-86), or dedifferentiated human articular chondrocytes (Liu, H. et al., 1998. Biochim Biophys Acta 1425:505-515) in alginate beads.
Yet another approach involves culturing rabbit growth plate chondrocytes in soft agar, or on a substrate coated with type I collagen, type II collagen or fibronectin (Enomoto-Iwamoto, M. et al., 1997. J Bone Miner Res 12:1124-1132).
Still another approach involves culturing primary chondrocytes in the presence of fibroblast growth factor (FGF)-2 in three-dimensional polymer scaffolds (Martin, I. et al., 1999. Exp Cell Res 253:681-688).
A further approach involves culturing dedifferentiated rabbit articular chondrocytes in the presence of transforming growth factor (TGF)-beta 1, with or without the microfilament modifying drug dihydrocytochalasin B (DHCB; Benya, P. D. and Padilla, S. R., 1993. Exp Cell Res 204:268-277).
Yet a further approach involves culturing primary chondrocytes (Borge, L. et al., 1997. In-vitro Cell Dev Biol Anim 33:703-709), mesenchyme of chick embryo wing bud in micromass cultures (Kulyk, W. M. et al., 2000. Exp Cell Res 255:327-332), or fibroblasts under hypoxic conditions (U.S. Pat. No. 6,489,165) in the presence of the protein kinase C (PKC)/actin polymerization antagonist staurosporine.
Still a further approach involves culturing mesenchymal progenitor cells using chemically defined components (U.S. Pat. Application No. 20030026786).
All of the aforementioned approaches, however, suffer from significant disadvantages including: incapacity to generate cultured cartilage/bone displaying optimal cartilage/bone specific differentiation, and/or displaying such differentiation for an optimally long duration in-vitro; and/or their being excessively cumbersome/complex and/or expensive to practice, such as in the case of approaches involving the use of three dimensional supports or biomolecule-coated substrates.
Thus, all prior art approaches have failed to provide an adequate solution for generating cultured cartilage/bone.
There is thus a widely recognized need for, and it would be highly advantageous to have, a method of generating cultured cartilage/bone devoid of the above limitation.